Multifaceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus during Hypoxia

点击翻译按钮获取中文摘要

 DESCRIPTION (provided by applicant): Hypoxia, or low oxygen (O2) levels, poses a great physiological challenge for many organisms, including aquatic invertebrates and mammals. Without proper O2 levels, cells are unable to respire, meaning glucose cannot be utilized, which can be detrimental to the cell. Lack of O2 is often seen in several medical conditions (e.g. asthma), although we lack a molecular understanding on the major biochemical changes due to hypoxia. Neuropeptides are thought to be a major class of regulators for these stress-induced responses; however, the full complement of neuropeptides and their expression changes during stress are not well characterized. This is mainly due to the complexity of the mammalian nervous system and current lack of tools to probe such intricate systems. Invertebrates (e.g. crustaceans), with their simple nervous system and well-characterized physiology, are an excellent model for understanding the roles of neuropeptides in response to stress. Crustaceans also contain several neuropeptide homologs to higher order animals, suggesting that this research can be easily translated to a mammalian model system. With the development of high resolution and mass accurate instrumentation, mass spectrometry (MS) has become the preferred technique to study neuropeptides. Also, the development of mass spectrometric imaging (MSI) has allowed for high-throughput analysis of molecular species in a biological tissue with no prior knowledge, thus obtaining the spatial information of hundreds of analytes in one experiment. With its high sensitivity and selectivity, MS will be used to quantitatively study the changes in the neuropeptidome due to the hypoxic stress response. Hence, I propose the following goals: 1) to investigate the in vivo expression changes of neuropeptides in the crustacean nervous system caused by a hypoxic environment using ESI- and MALDI-MS, 2) to analyze the neuropeptide distribution changes due to hypoxia exposure via MALDI-MSI, and 3) to develop a method to determine absolute quantities of neuropeptides via MALDI-MSI with custom multiplexed mass difference isotopic tags (iDiLeu). Collectively, these aims will develop MS as an enabling analytical tool to facilitate the understanding of the molecular mechanisms underlying the regulation of hypoxia inside the crustacean nervous system, which will give us novel insight into how the mammalian nervous system changes due to hypoxic conditions.